Study On The Inhibition Effect Of Ag Nanoparticles On Nosema Bombycis

Microsporidia is a class of obligate intracellular parasitic single-cell eukaryotic microorganisms,which are widely distributed and hosted by almost all invertebrates and vertebrates including humans.Nosema bombycis(N.b)is the first microsporidia that be identified.It can infect Bombyx mori and cause Pébrine,causing significant economic losses to the silkworm industry.At present,the main methods of preventing and controlling Pébrine include: female moth microscopic examination to eliminate toxic silkworm species,strict disinfection during feeding,and treatment with chemical drugs such as fangweiling and albendazole after infection.Due to the special structure of N.b,the effects of some therapeutic drugs are still unstable.The prevention and control of N.b has been based on prevention.However,the existing preventive measures are also difficult to achieve an ideal effect,new and efficient methods are urgently needed.Ag nanoparticles(AgNPs)are metallic silver with a nanometer size.Due to its unique surface effects,volume effects,quantum size effects and macro tunneling effects,AgNPs have been widely used in chemical,textile,electronics,life science and medical research fields,and presents extremely important application value.AgNPs have broad-spectrum antibacterial properties,long-lasting antibacterial effects,and hard to produce drug resistance.It can effectively kill pathogenic microorganisms such as bacteria,fungi and mycoplasma.In this study,in order to investigate whether AgNPs have an inhibitory effect on silkworm pathogenic microorganisms,especially N.b,and the mechanism of action on N.b,relevant research was conducted and the following research results were obtained: 1.Effect of AgNPs on growth and development of silkwormThe properties of AgNPs was observed by transmission electron microscopy,AgNPs was spherical,with a size of 3~24 nm,mainly distributed in 3~18 nm,and the lattice fringe spacing was 0.23 nm.Cell proliferation activity experiments showed that AgNPs did not affect the proliferation activity of BmN-SWU1 cell with concentration below 12 ?g/m L.Further select five concentrations of AgNPs at 1 ?g/m L,10 ?g/mL,25 ?g/mL,50 ?g/mL and 100 ?g/mL to test the toxicity on the silkworm larvae.The results showed that there were no significant differences in the weight of silkworm larvaes,the cocoon weight,the cocoon shell weight,the cocoon shell rate and the number of eggs laid between the groups,indicating that the AgNPs were safe for silkworm larvae with concentration below 100 ?g/m L.2.Inhibitory effect of AgNPs on the main pathogens of silkwormThe pathogenic microorganisms of silkworm include virus,bacteria and fungi.Among them,Bombyx mori nucleopolyhedrovirus(BmNPV)and N.b have the greatest threat to silkworm.In this study,BmNPV,N.b and several representative bacterias were selected as the research object of AgNPs to inhibit the pathogenic microorganisms of silkworm.First,the inhibitory effect of AgNPs on BmNPV was identified.At the individual level,qRT-PCR analysis found that the expression of viral genes remained at a low level after the AgNPs treatment.The statistical analysis of survival rate showed that the AgNPs treatment significantly improved the survival rate of silkworm.The results of AgNPs inhibitory effect on silkworm pathogenic bacterias showed that AgNPs can completely inhibit the growth and reproduction of Serratia marcescens,Bacillus thuringiensis berliner and Bacillus sp with the concentration above 25 ?g/m L.Furthermore,the effect of AgNPs on inhibiting N.b was tested.Statistics on the survival rate of silkworms infected with N.b found that AgNPs can increase the survival rate of silkworms.After the effect of 100 ?g/m L of AgNPs,the survival rate of silkworms reached 90% on the tenth day.At this time,the survival rate of silkworm normally infected with N.b was only 20%.The results of qRT-PCR analysis found that the copy number of N.b was lower than that of the control group,and significantly decreased with the increase of AgNPs concentration.It was proved that AgNPs could inhibit the proliferation of N.b.The above results indicate that AgNPs have inhibitory effects on BmNPV,silkworm bacteria and N.b,have broad-spectrum resistance to pathogenic microorganisms of silkworm.3.Study on the mechanism of Ag NPs effect on Nosema bombycisBased on the current status of the prevention and control of pathogenic microorganisms in silkworm,N.b was selected as the main research object to explore the mechanism of AgNPs effecting on N.b.After artificially germinating,it was found that the germination rate of the N.b was decreased with the increased concentration of AgNPs,so that the infectivity of the N.b was affected.Observing the structure of the N.b by transmission electron microscopy,it was found that after the AgNPs treatment,the exospore became smooth,the endospore with a transparent electron layer became thicker,and the internal structure of the spores was extremely disordered,such as nuclei,organelles,germination devices,etc.Furthermore,through scanning electron microscope observation,it was found that AgNPs quickly acted on the spore wall after contacting the N.b.With the extension of time,a portion of N.b cracked and shrivelled.The nuclei of N.b treated with AgNPs were stained and qRT-PCR detected,and the nuclear material was found to be released.The total protein detection results of N.b showed that AgNPs treatment caused the N.b to release a large amount of protein,which caused the protein is severely denatured and degraded.The above results indicate that AgNPs will seriously damage the structure of N.b,and interact with the spore wall to increase its permeability,so that some of the damaged DNA and protein and other substances are released,which greatly weakens the ability to infect and reduces the pathogenic ability of N.b.